The evolution of dengue-2 viruses in Malindi, Kenya and greater East Africa: Epidemiological and immunological implications.

Kenya experiences a substantial burden of dengue, yet there are very few DENV-2 sequence data available from this country and indeed the entire continent of Africa. We therefore undertook whole genome sequencing and evolutionary analysis of fourteen dengue virus (DENV)-2 strains sampled from Malindi sub-County Hospital during the 2017 DENV-2 outbreak in the Kenyan coast. We further performed an extended East African phylogenetic analysis, which leveraged 26 complete African env genes. Maximum likelihood analysis showed that the 2017 outbreak was due to the Cosmopolitan genotype, indicating that this has been the only confirmed human DENV-2 genotype circulating in Africa to date. Phylogeographic analyses indicated transmission of DENV-2 viruses between East Africa and South/South-West Asia. Time-scaled genealogies show that DENV-2 viruses shows spatial structure at the country level in Kenya, with a time-to-most-common-recent ancestor analysis indicating that these DENV-2 strains were circulating for up to 5.38 years in Kenya before detection in the 2017 Malindi outbreak. Selection pressure analyses indicated sampled Kenyan DENV strains uniquely being under positive selection at 6 sites, predominantly across the non-structural genes, and epitope prediction analyses showed that one of these sites corresponds to a putative predicted MHC-I CD8+ DENV-2 Cosmopolitan virus epitope only evident in a sampled Kenyan virus. Taken together, our findings indicate that the 2017 Malindi DENV-2 outbreak arose from a strain which had circulated for several years in Kenya before recent detection, has experienced diversifying selection pressure, and may contain new putative immunogens relevant to vaccine design. These findings prompt further genomic epidemiology studies in this and other Kenyan locations to further elucidate the transmission dynamics of DENV in this region.

Adenosine, AMP, and protein phosphatase activity in sandfly saliva.

As they probe the skin for blood, sand flies inject saliva that prevents hemostasis. Sand fly saliva also promotes leishmaniasis by suppressing immunologic functions of macrophages. Saliva of Phlebotomus papatasi, the vector of Old World cutaneous leishmaniasis, contains adenosine and AMP. We show that Ph. papatasi saliva as well as pure adenosine down-regulate the expression of the inducible nitric oxide (NO) synthase gene in activated macrophages. In addition Ph. papatasi, but not Lutzomyia longipalpis, saliva inhibits the production of NO. Taken together, these data suggest that salivary adenosine is responsible for the down-regulation of NO synthesis. Saliva of both genera Phlebotomus and Lutzomyia contains significant levels of endogenous protein phosphatase-1/2A-like activity that is heat labile, inhibitable by okadaic acid and calyculine a, and does not require divalent cations.

Red cell surface changes and erythrophagocytosis in children with severe plasmodium falciparum anemia.

Severe anemia is one of the most lethal complications in children infected with Plasmodium falciparum. The pathogenesis of this anemia is not completely understood. Experimental data from malaria-infected humans and animal models suggest that uninfected red cells have a shortened life span. This study looked for changes in the red cell surfaces of children with severe malarial anemia that could explain this accelerated destruction. A prospective case-control study was conducted of children with severe P falciparum anemia (hemoglobin of 5 g/dL or lower) admitted to a large general hospital in western Kenya. Children with severe anemia were compared with children who had symptoms of uncomplicated malaria and with asymptomatic children. Cytofluorometry was used to quantify in vitro erythrophagocytosis and to measure red cell surface immunoglobulin G (IgG) and the complement regulatory proteins CR1, CD55, and CD59. Red cells from patients with severe anemia were more susceptible to phagocytosis and also showed increased surface IgG and deficiencies in CR1 and CD55 compared with controls. Red cell surface CD59 was elevated in cases of severe anemia compared with asymptomatic controls but not as compared with symptomatic controls. The surface of red cells of children with severe P falciparum anemia is modified by the deposition of IgG and alterations in the levels of complement regulatory proteins. These changes could contribute to the accelerated destruction of red cells in these patients by mechanisms such as phagocytosis or complement-mediated lysis. (Blood. 2000;95:1481-1486)

Glucose uptake in Trypanosoma vivax and molecular characterization of its transporter gene.

A gene, TvHT1, encoding a glucose transporter protein, has been cloned from the haemoflagellate protozoon, Trypanosoma vivax, which has an active Kreb's cycle in the mammalian stage. The deduced polypeptide is similar in amino acid sequence to other kinetoplastid hexose transporters from Trypanosoma brucei (THT1 and THT2), Trypanosoma cruzi (TcrHT1) and Leishmania (Pro-1). The similarity is higher with THT2 (expressed in T. brucei insect forms) than with the other isoforms. The kinetic properties of glucose uptake in Chinese Hamster Ovary (CHO) cells expressing TvHT1 and in trypanosomes show s a saturable transport mechanism typical of a facilitated carrier system, with a similar affinity for D-glucose as that of the T. brucei bloodstream form carrier, THT1 (Km = 0.548 +/- 0.01 mM, Vmax = 4.26 +/- 0.12 nmol.min-1.mg protein-1 in CHO cells and Km = 0.585 +/- 0.068 mM, Vmax = 88.5 +/- 6.2 nmol.min-1.mg protein-1 in T. vivax). The specificity of the TvHT1 protein for various D-glucose analogues, as judged by inhibition of 2-deoxy-D-arabinose-hexose transport, shows properties that are intermediate between those of THT1 on the one hand and TcrHT1 and THT2 on the other. As with the hexose transporters in the other members of Kinetoplastida, the TvHT1-encoded system differs from erythrocyte-type glucose transport by its moderate sensitivity to cytochalasin B and its capacity to transport fructose.

Electrophoretic karyotyping is a sensitive epidemiological tool for studying Trypanosoma evansi infections.

Thirty-six isolates of trypanozoon trypanosomes collected from camels in Northern Kenya during the dry season sporadic infections of 1986 and during the wet season epidemic infections of 1987 were identified as Trypanosoma evansi by the homogeneity of their kinetoplast DNA minicircles. Although the minicircles of all the isolates were indistinguishable, polymorphism in chromosome-sized DNA molecules detected by electrophoresis was extensive. The isolates could be grouped into eight distinct electrophoretic karyotypes which could be distinguished from three additional karyotypes identified among earlier T. evansi isolates. In one camel herd with a long history of trypanocide application, which was continued during the present study, all isolates bar one belonged to one karyotype group. From a second herd, in which trypanosomosis management was by individual treatment of proven parasitaemic cases, isolates with diverse karyotypes were obtained. Some of the karyotypes identified during the dry season sporadic infections were re-isolated in the subsequent wet season epidemic. These observations indicate that distinguishing T. evansi isolates by molecular electrophoretic karyotypes is more discriminating than kDNA analysis. Observations of karyotype patterns recurring in isolates from herds kept under chemoprophylaxis could help in the identification of drug-resistant parasites.

A comparison of the antigen detection ELISA and parasite detection for diagnosis of Trypanosoma evansi infections in camels.

Two herds of 60 camels each, living in Trypanosoma evansi endemic areas, were selected and studied for a period of 18 months. Animals in one herd were treated prophylactically with quinapyramine prosalt (May and Baker, Dagenham, UK), while those in the other herd were treated individually with quinapyramine dimethylsulphate (May and Baker, Dagenham, UK) when proven parasitaemic. The herd on prophylaxis was sampled for antigen and patent infection monthly. The other herd was sampled weekly for patent infection and fortnightly for antigen. The results obtained could be divided into four categories. The first category comprised cases (52 out of 61) in which the presence of trypanosome antigens could be correlated with parasitological diagnosis. In 80% of these animals the antigens disappeared from the circulation within a period of 30 days following chemotherapy. The second category comprised those animals with parasitologically proven infections but which did not have antigens in their sera. This was observed in nine camels, seven of which were from the herd that was being examined weekly for the presence of trypanosomes. These were considered to be animals in early infection, as the subsequent sera were also negative for anti-trypanosome antibodies and immune complexes. The third category comprised camels which were antigen-positive but aparasitaemic. Sera from these animals were also positive for anti-trypanosome antibodies, indicating that antigen-positivity was a true reflection of trypanosome infections in these animals. The last category comprised pre-weaned camel calves which appeared to have some form of protection against trypanosomiasis, as evidenced by the absence of trypanosomes, antigens and antibodies throughout the early period of their lives. Only occasional antigenaemia was found in a few calves. It is concluded that trypanosome antigen detection may give a more accurate idea of the prevalence of T. evansi infections than does whole parasite detection.

Trypanosoma (Nannomonas) congolense: molecular characterization of a new genotype from Tsavo, Kenya.

Trypanosoma (Nannomonas) congolense comprises morphologically identical but genetically heterogeneous parasites infective to livestock and other mammalian hosts; three different genotypes of this parasite have been described previously. Restriction enzyme fragment length polymorphisms (RFLPs) in both kinetoplast DNA minicircle and nuclear DNA sequences, and randomly amplified polymorphic deoxyribonucleic acid (RAPD) patterns have been used here to demonstrate the existence of another type of T. (N.) congolense that is genotypically distinct from those that have so far been characterized at the molecular level. A highly repetitive, tandemly arranged DNA sequence and oligonucleotide primers, for use in polymerase chain reaction (PCR) amplification are described, which can be used for specific identification of the trypanosome and its distinction from others within the Nannomonas subgenus.

Serodiagnosis of African sleeping sickness in vervet monkeys by detection of parasite antigens.

Sera of vervet monkeys experimentally infected with T. b. rhodesiense were examined using a double antibody sandwich ELISA and Procyclic Agglutination Trypanosomiasis Test (PATT) for the presence of circulating trypanosomal antigens and anti-procyclic surface antibodies, respectively. Trypanosomal antigens were detected at 7 days post infection and remained at a detectable level thereafter during the infection. Antigens were not detected in sera prior to experimental infection or at 26 days after trypanocidal drug treatment. Although both the PATT and the sandwich ELISA results correlated with the infection status of the animals, the sandwich ELISA gave a better indication of the disease progression than the PATT, especially during trypanocidal drug therapy. The results illustrate the potential utility of the double antibody sandwich ELISA for diagnosis of African sleeping sickness.

The relapse of Trypanosoma brucei brucei infections after chemotherapy in rabbits.

Infections of T. brucei in the rabbit were found to relapse after chemotherapy. The results indicated that 25 mg/kg diminazene aceturate given 3 days after infection resulted in a complete cure but if given 7 days after infection relapses frequently occurred. However, treatment was apparently successful if delayed until 14 or 21 days. Six of the rabbits originally treated with diminazene aceturate on day 7 were treated with suramin 21 days later; in 3 rabbits the infections relapsed. In all rabbits in which drug treatment was not curative, the clinical condition nevertheless improved. An attempt to locate a cryptic focus of infection in rabbits was unsuccessful.

Evidence of blood cerebrospinal fluid barrier permeability impairment in Trypanosoma rhodesiense infection in vervet monkeys.

Blood-cerebrospinal fluid (CSF) barrier damage in 11 vervet monkeys was estimated before infection and during the early and late phases of Trypanosoma rhodesiense disease, using the method given by Tibbling, Link and Ohman (1977). Of the 11, only one monkey showed signs of barrier impairment that ranged from a slight (12.6) to total barrier impairment (285); the latter occurring just before the height of clinical encephalitis. The barrier reverted to normal after melarsoprol treatment at 1.8 mg/kg X 4. Between the two extremes of barrier impairment, there were periods of remission during which time the barrier reverted to normal. This monkey however continued to suffer epileptic fits for a period of three months, a condition that started soon after the treatment.

Use of procyclic trypanosomes for detection of antibodies in sera from vervet monkeys infected with Trypanosoma rhodesiense: an immunodiagnostic test for African sleeping sickness.

Uncoated procyclic culture forms of African trypanosomes were used in immunofluorescence and simple agglutination assays to detect antibodies in the sera of vervet monkeys infected with T. b. rhodesiense. Antibodies to procyclic surface antigens were found in sera from animals with active, untreated infections or sera taken soon after treatment with trypanocidal drugs. The antibodies were detectable within 7 days of infection. No specific antibodies were detected in sera prior to infection or long after drug cure. The results indicate that antigens expressed on the surface of procyclic culture forms of T. brucei spp. are useful for the detection of antibodies produced in response to infection with T. b. rhodesiense and may allow the development of a simple immunodiagnostic test for African sleeping sickness. In addition, the use of a form of the trypanosome of a different differentiation state from the infecting organism illustrates the utility of this approach for detection of antibodies to common antigens.